Peyer&#39;s patch and/or M-cell targeting ligands

ABSTRACT

Purified synthetic polypeptide ligands for targeting pharmaceutical agents and carriers comprising such agents to intestinal epithelial tissue, especially Peyer&#39;s patch and/or M-Cell tissue. Also methods of using the ligands.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application60/302,591 filed Jul. 2, 2001, which application is incorporated byreference herein in its entirety.

FIELD OF INVENTION

This invention relates to novel targeting ligands which permit orfacilitate the transport of drugs, macromolecules, or particles, such asbiodegradable nanoparticles and microparticles, or bacterial carriers orviral carriers through the intestinal epithelium, M-cells located in gutassociated lymphoid tissue, and/or Peyer's Patch tissue of theintestinal epithelium.

BACKGROUND OF THE INVENTION

The epithelial cells lining the lumenal side of the gastrointestinaltract (GIT) are a major barrier to drug delivery following oraladministration. However, there are four recognized transport pathwayswhich can be exploited to facilitate drug delivery and transport: thetranscellular, paracellular, carrier-mediated and transcytotic transportpathways. The ability of a conventional drug, peptide, protein,macromolecule, nanoparticulate system or microparticulate system tointeract with one of these transport pathways may result in increaseddelivery of that drug or particle from the GIT to the underlyingcirculation.

M-cells are antigen sampling cells that are found in the epithelium ofthe gut-associated lymphoid tissue, or Peyer's Patch. The transcytoticcapacity of M-cells and the downstream processing of the antigen sampledwould suggest that targeting vaccines to M-cells would enhance oralimmunization (Foster et al., 15 Vaccine 546-71 (1998)). However, todate, no human M-cell marker has been identified as a target fordelivery of vaccines and/or other drugs through the M-cell route.

In U.S. Pat. No. 6,117,632 to O'Mahony, one of the present inventorsdisclosed a method of identifying peptides which permit or facilitatethe transport of an active agent through human or animal epithelialtissue. This method uses in vivo phage display screening to identifyligands.

U.S. Pat. No. 6,060,082 to Chen et al. discloses modified polymerizedliposomes that contain a molecule or ligand on their surfaces in orderto target the liposomes to a specific site or cell type for oral/mucosaldrug delivery. Also disclosed is an embodiment in which the liposomesare modified with carbohydrate moities or lectins that specificallytarget M-cells or Peyer's Patches in mice. However, this reference onlyteaches transport of liposomes.

Other approaches include: drug delivery through the epithelium by acarrier molecule selected from transferrin receptor ligands conjugatedto an active agent and a transport enhancing agent (U.S. Pat. No.5,254,342 to Shen et al.); and coupling the antigen to ligands that bindthe FcRn receptor. (U.S. Pat. No. 6,030,613 to Blumberg et al.).

All references cited herein are incorporated herein by reference intheir entireties.

Thus, there still exists a need for M-cell and/or Peyer's Patch specificligands that are particularly effective in transporting drugs, includingdrug-loaded nanoparticles and microparticles, or bacterial or viralcarries coding for vaccines into or across a human or animal intestinalepithelium.

BRIEF SUMMARY OF THE INVENTION

In an aspect directly related to specific 12-mer L-peptides, theinvention is a purified synthetic polypeptide ligand comprising a 12-merL-peptide, fragment or homologue thereof, said 12-mer L-peptide selectedfrom the group consisting of SEQ ID NOs:1-34, SEQ ID NOs: 38-39, and SEQID NO:42 wherein said fragment is at least five contiguous amino acidsand wherein said homologue is at least 9/12 homologous to a 12-merpeptide selected from said group and wherein said 12-mer L-peptide,fragment or homologue thereof, when integrated as an N-terminal PIIIfusion peptide of an M13 phage confers an ability to bind the phage toeither Caco-2 cell, IEC-6 cell, rat, mouse, pig or dog homogenatemembrane fractions, said ability being at least as great as thatconferred by a similarly integrated 12-mer peptide of SEQ ID NO:67.

The above noted functional test is “wherein said 12-mer L-peptide,fragment or homologue thereof, when integrated as an N-terminal PIIIfusion peptide of an M13 phage confers an ability to bind the phageeither to Caco-2 cell, IEC-6 cell, rat, mouse, pig or dog homogenatemembrane fractions, said ability being at least as great as thatconferred by a similarly integrated 12-mer peptide of SEQ ID NO:67”, andis also specified below for other aspects and embodiments of theinvention. For all agents and embodiments, preferred ligands are thosethat satisfy the functional test when Caco-2 cell homogenate membranefractions are used.

“9/12 homologous to a 12-mer peptide” means that if one aligns ahomologue with the 12-mer peptide, 9 of 12 amino acids, contiguous ornot, are identical to the 12-mer peptide. For example, if a peptidecontained the sequence LTPPPWLVRTRP, it would be 9/12 homologous to the12-mer peptide of SEQ ID NO:1 (ATPPPWLLRTAP).

It follows that, in the above noted aspect of the invention a peptidecan be 9/12 homologous to a specified 12-mer peptide, but not a fragmentof at least five contiguous amino acids of that 12-mer peptide.Conversely, a peptide can be a fragment of at least five contiguousamino acids (e.g. 5-8 amino acids), but not at least 9/12 homologous tothe 12-mer peptide. However, a peptide can also be both: a fragment ofat least five amino acids of the 12-mer peptide and at least 9/12homologous to the 12-mer peptide. A peptide that is 9/12 homologous to a12-mer peptide is 75% homologous to that peptide.

In an aspect related to the D-forms of the specific 12-mer L-peptides,the invention is a purified synthetic polypeptide ligand comprising a12-mer D-peptide, fragment or homologue thereof, said 12-mer D-peptidebeing the D-form of a 12-mer L-peptide selected from the groupconsisting of D-forms of 12-mer L-peptides of SEQ ID NOs:1-34, SEQ IDNOs:38-39 and SEQ ID NO:42, wherein said fragment is at least fivecontiguous amino acids and wherein said homologue is at least 9/12homologous to a 12-mer D-peptide selected from said group and whereinsaid 12-mer D-peptide, fragment or homologue, when integrated as anN-terminal PIII fusion peptide of an M13 phage confers an ability tobind the phage to either Caco-2 cell, IEC-6 cell, rat, mouse, pig or doghomogenate membrane fractions, said ability being at least as great asthat conferred by a similarly integrated 12-mer peptide of SEQ ID NO:67.

In an aspect related to the retro-inverted forms of the specific 12-merL-peptides, the invention is a purified synthetic polypeptide ligandcomprising a 12-mer retro-inverted peptide, fragment or homologuethereof, said 12-mer retro-inverted peptide being the retro-invertedform of a 12-mer L-peptide selected from the group consisting ofretro-inverted forms of 12-mer L-peptides of SEQ ID NOs:1-34, SEQ IDNOs:38-39 and SEQ ID NO:42, wherein said fragment is at least fivecontiguous amino acids and wherein said homologue is at least 9/12homologous to a 12-mer retro-inverted peptide selected from said groupand wherein said 12-mer retro-inverted peptide, fragment or homologue,when integrated as an N-terminal PIII fusion peptide of an M13 phageconfers an ability to bind the phage to either Caco-2 cell, IEC-6 cell,rat, mouse, pig or dog homogenate membrane fractions, said ability beingat least as great as that conferred by a similarly integrated 12-merpeptide of SEQ ID NO:67.

In an aspect related to specific peptide motifs, the invention is apurified synthetic polypeptide ligand, said ligand comprising aL-peptide motif, D-peptide version thereof, or retro-inverted versionthereof, said L-peptide motif being selected from the group consistingof TPPP, PPY, PVT, LGT, NVY, HESSH (SEQ ID NO:97) and NVYTXXXXSPXP (SEQID NO:98), wherein said L-peptide motif, a D-peptide version thereof, ora retro-inverted version thereof when integrated as an N-terminal PIIIfusion peptide of an M13 phage confers an ability to bind the phage toeither Caco-2 cell, IEC-6 cell, rat, mouse, pig or dog homogenatemembrane fractions, said ability being at least as great as thatconferred by a similarly integrated 12-mer peptide of SEQ ID NO:67.

In an aspect related to naturally occurring homologues of the specificL-peptides, the invention is a purified synthetic polypeptide ligand,not more than 200 amino acids in length, comprising an L-peptide,fragment or homologue thereof, said L-peptide being 6 to 12 amino acidsin length, and said L-peptide being selected from the group consistingof SEQ ID NOs:74 through SEQ ID NO:96, wherein said fragment is at leastfive contiguous amino acids and wherein said homologue is at least 83%homologous to an L-peptide selected from said group wherein saidL-peptide, fragment or homologue thereof when integrated as anN-terminal PIII fusion peptide of an M13 phage confers an ability tobind the phage to either Caco-2 cell, IEC-6 cell, rat, mouse, pig or doghomogenate membrane fractions, said ability being at least as great asthat conferred by a similarly integrated 12-mer peptide of SEQ ID NO:67.

In an aspect related to the D-forms of naturally occurring homologues ofthe specific L-peptides, the invention is a purified syntheticpolypeptide ligand, not more than 200 amino acids in length, comprisinga D-peptide, fragment or homologue thereof, said D-peptide being 6 to 12amino acids in length and said D-peptide being the D-form of a L-peptideselected from the group consisting of SEQ ID NOs:74 through SEQ IDNO:96, wherein said fragment is at least five contiguous amino acids andwherein said homologue is at least 83% homologous to a D-peptideselected from said group and wherein said D-peptide, fragment orhomologue thereof when integrated as an N-terminal PIII fusion peptideof an M13 phage confers an ability to bind the phage to either Caco-2cell, IEC-6 cell, rat, mouse, pig or dog homogenate membrane fractions,said ability being at least as great as that conferred by a similarlyintegrated 12-mer peptide of SEQ ID NO:67.

In an aspect related to the retro-inverted forms of the naturallyoccurring homologues of the specific L-peptides, the invention is apurified synthetic polypeptide ligand, not more than 200 amino acids inlength, comprising a retro-inverted peptide, fragment or homologuethereof, said retro-inverted peptide being 6 to 12 amino acids in lengthand said retro-inverted peptide being the retro-inverted form of aL-peptide selected from the group consisting of SEQ ID NOs:74 throughSEQ ID NO:96, wherein said fragment is at least five contiguous aminoacids and wherein said homologue is at least 83% homologous to aretro-inverted peptide wherein said retro-inverted peptide, fragment orhomologue thereof when integrated as an N-terminal PIII fusion peptideof an M13 phage confers an ability to bind the phage to either Caco-2cell, IEC-6 cell, rat, mouse, pig or dog homogenate membrane fractions,said ability being at least as great as that conferred by a similarlyintegrated 12-mer peptide of SEQ ID NO:67.

In the above inventions, related to purified synthetic polypeptideligands there are preferred and even highly preferred embodiments.

As regards the inventions related to the specific 12-mer L-peptides orthe naturally occurring 12-mer homologues, their D-forms and theirretro-inverted forms, it is preferred that the homologue is at least10/12 homologous, more preferably 11/12 homologous. Similarly it ispreferred that fragments of a 12-mer be at least 8 amino acids inlength. Most preferred is the presence of the intact specific L-peptide,D-form, or retro-inverted form, rather than a homologue or fragment.

As to all of the aforementioned purified synthetic polypeptide ligands,it is preferred that their length be not more than 200 amino acids, morepreferably not more than 100 amino acids, most preferably not more than50 amino acids. Conversely, it is preferred that their length be atleast 12 amino acids, more preferably at least 20 amino acids, mostpreferably at least 30 amino acids.

In particular embodiments of all of the aforementioned purifiedsynthetic polypeptide ligands, the polypeptide comprises a zinc-bindingdomain.

Nucleic acid molecules that code for the aforementioned purifiedsynthetic polypeptide ligands are also aspects of the invention.Preferred are those that are not more than 600 nucleotides in length.Highly preferred are those that code for a purified syntheticpolypeptide that comprises one of the specific 12-mer peptides, motifs,or naturally occurring homologues.

In particular embodiments of the invention, one of the aforementionedpurified synthetic polypeptides ligands is integrated into the proteinof a phage.

In particular embodiments of the invention, one of the aforementionedpurified synthetic polypeptide ligands is covalently or non-covalentlybound to a carrier entity comprising a pharmaceutical agent. Forexample, the carrier entity is selected from the group consisting of ananoparticle, microparticle, liposome, bacterium, phage (bacteriophage)and s virus (preferably a mammalian virus, most preferably a humanvirus; especially non-pathogenic forms made by recombinant or othertechnologies). It is preferred that the nanoparticle, microparticle orliposome have a largest dimension that is in the range of 10 nm to 500μm, as discussed in more detail elsewhere herein. In particularembodiments of the invention, the pharmaceutical agent is a drug ortherapeutic agent. In other specific embodiments, the pharmaceuticalagent is a pathogen antigen.

Certain aspects of the invention involve the use of the purifiedsynthetic polypeptide ligands to target delivery of pharmaceuticalagents.

In one aspect, the invention is a method of administering apharmaceutical agent to an organism having intestinal epithelium, saidmethod comprising contacting said intestinal epithelium with one of theaforementioned purified synthetic polypeptide ligands that iscovalently, or non-covalently bound to, a carrier entity. In preferredthe embodiments, the organism is a mammal. Most preferably, the mammalis a human.

In particular embodiments of the method, the carrier entity is selectedfrom the group consisting of a nanoparticle, microparticle, liposome,bacterium, phage and virus. A preferred embodiment is where thepolypeptide ligand is expressed on the surface of a phage or bacteriumfurther comprising an antigen or a gene encoding the antigen alsoexpressed on the surface.

Preferably, the microparticle, nanoparticle or liposome has its majordimension in the range of 10 nm to 500 μm. In preferred embodiments, thecarrier entity is loaded with a pharmaceutical agent. The preferredroute of administration for delivery of the ligand-carrier entity is theoral route. Other possible routes are the rectal, subcutaneous,intramuscular, nasal and intravenous routes. In particular embodimentsthe purified synthetic polypeptide ligand is a 12-mer integrated into acoat protein of a phage. In other particular embodiments, the purifiedsynthetic polypeptide ligand comprises a zinc-binding motif, and saidligand is contacted with said epithelium in the presence of zinc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of the binding of the phage from Rat 1 to rat Peyer'spatch tissue as a function of absorbance at 405 nm;

FIG. 1B is a graph of the binding of the phage from Rat 1 to rat Peyer'spatch tissue as a function of absorbance at 405 nm;

FIG. 2 is a graph of the binding of the phage from Rat 2 to rat Peyer'spatch tissue as a function of absorbance at 405 nm;

FIG. 3 is a graph of the binding of the phage from Rat 3 to rat Peyer'spatch tissue as a function of absorbance at 405 nm;

FIG. 4 is a graph of the binding of the phage from Rat 4 to rat Peyer'spatch tissue as a function of absorbance at 405 nm;

FIG. 5 is a graph of the binding of the phage form Rat 5 to rat Peyer'spatch tissue as a function of the absorbance at 405 nm;

FIG. 6 is a graph of the binding of the 55 high binding clones to ratsmall intestinal homogenates with and without Peyer's patch tissuepresent as a function of absorbance at 405 nm;

FIG. 7 is a graph of the binding of the remaining 55 high binding clonesto rat small intestinal homogenates with and without Peyer's patchtissue present as a function of absorbance at 405 nm;

FIG. 8 is a graph of the binding of the clones to dog small intestinalhomogenates with and without Peyer's patch tissue present as a functionof absorbance at 405 nm;

FIG. 9 is a graph of the binding of the clones to pig small intestinalhomogenates with and without Peyer's patch tissue present as a functionof absorbance at 405 nm;

FIG. 10 is a graph of the binding of the clones to mouse smallintestinal homogenates with and without Peyer's patch tissue present asa function of absorbance at 405 nm;

FIG. 11 is a graph of the binding of the phage clones to rat Peyer'spatch, Caco-2 cells and IEC-6 cells as a function of absorbance at 405nm;

FIG. 12 is a graph of the binding of the phage clones to differentiatedand non-differentiated Caco-2 cells as a function of absorbance at 405nm;

FIG. 13A is a graph of the binding of SEQ ID NO:3 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13B is a graph of the binding of SEQ ID NO:4 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13C is a graph of the binding of SEQ ID NO:7 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13D is a graph of the binding of SEQ ID NO:8 with a biotin tag atthe amino and carboxyl terminal end to Caco-2 homogenates and rat GIhomogenates as a function of absorbance at 650 nm. Results were obtainedusing an ELISA-based assay with streptavidin-peroxidase detection.

FIG. 13E is a graph of the binding of SEQ ID NO:5 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13F is a graph of the binding of SEQ ID NO:14 with a biotin tag atthe amino and carboxyl terminal end to Caco-2 homogenates and rat GIhomogenates as a function of absorbance at 650 nm. Results were obtainedusing an ELISA-based assay with streptavidin-peroxidase detection.

FIG. 13G is a graph of the binding of SEQ ID NO:9 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13H is a graph of the binding of SEQ ID NO:20 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13I is a graph of the binding of SEQ ID NO:17 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13J is a graph of the binding of SEQ ID NO:28 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm.

Results were obtained using an ELISA-based assay withstreptavidin-peroxidase detection.

FIG. 13K is a graph of the binding of SEQ ID NO:26 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm.

Results were obtained using an ELISA-based assay withstreptavidin-peroxidase detection.

FIG. 13L is a graph of the binding of SEQ ID NO:11 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with is streptavidin-peroxidase detection.

FIG. 13M is a graph of the binding of SEQ ID NO:10 with a biotin tag atthe amino terminal end to Caco-2 homogenates as a function of absorbanceat 650 nm. Results were obtained using an ELISA-based assay withstreptavidin-peroxidase detection.

FIG. 13N is a graph of the binding of SEQ ID NO:16 with a biotin tag atthe amino terminal end to Caco-2 homogenates as a function of absorbanceat 650 nm. Results were obtained using an ELISA-based assay withstreptavidin-peroxidase detection.

FIG. 130 is a graph of the binding of SEQ ID NO:19 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13P is a graph of the binding of SEQ ID NO:29 with a biotin tag atthe amino terminal end to Caco-2 homogenates and rat GI homogenates as afunction of absorbance at 650 nm. Results were obtained using anELISA-based assay with streptavidin-peroxidase detection.

FIG. 13Q is a graph of the binding of SEQ ID NO:30 with a biotin tag atthe amino terminal end to Caco-2 homogenates as a function of absorbanceat 650 nm. Results were obtained using an ELISA-based assay withstreptavidin-peroxidase detection.

FIG. 13R is a graph of the binding of SEQ ID NO:15 with a biotin tag atthe amino terminal end to Caco-2 homogenates as a function of absorbanceat 650 nm. Results were obtained using an ELISA-based assay withstreptavidin-peroxidase detection.

FIG. 14A is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at both the amino and carboxyl terminal end to pig GI homogenate asa function of absorbance at 650 nm.

FIG. 14B is a graph of the binding of SEQ-ID NOs: 8 and 14 with a biotintag at both the amino and carboxyl terminal end to pig Peyer's Patchhomogenate as a function of absorbance at 650 nm.

FIG. 14C is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at both the amino and carboxyl terminal end to rat GI homogenate asa function of absorbance at 650 nm.

FIG. 14D is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at both the amino and carboxyl terminal end to rat Peyer's patchhomogenate as a function of absorbance at 650 nm.

FIG. 14E is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at both the amino and carboxyl terminal end to dog GI homogenate asa function of absorbance at 650 nm.

FIG. 14F is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at both the amino and carboxyl terminal end to dog Peyer's patchhomogenate as a function of absorbance at 650 nm.

FIG. 14G is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at both the amino and carboxyl terminal end to mouse GI homogenateas a function of absorbance at 650 nm.

FIG. 14H is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at both the amino and carboxyl terminal end to mouse Peyer's patchhomogenate as a function of absorbance at 650 nm.

FIG. 14I is a graph of the binding of SEQ ID NOs: 3, 7 and 9 with abiotin tag at the amino terminal end to dog GI homogenate as a functionof absorbance at 650 nm.

FIG. 14J is a graph of the binding of SEQ ID NOs: 3, 7 and 9 with abiotin tag at the amino terminal end to dog Peyer's patch homogenate asa function of absorbance at 650 nm.

FIG. 14K is a graph of the binding of SEQ ID NOs: 3, 7 and 9 with abiotin tag at the amino terminal end to mouse GI homogenate as afunction of absorbance at 650 nm.

FIG. 14L is a graph of the binding of SEQ ID NOs: 3, 7 and 9 with abiotin tag at the amino terminal end to mouse Peyer's patch homogenateas a function of absorbance at 650 nm.

FIG. 15A is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at the amino and carboxyl terminal end to rat liver tissuehomogenate as a function of absorbance at 650 nm.

FIG. 15B is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at the amino and carboxyl terminal end to rat spleen tissuehomogenate as a function of absorbance at 650 nm.

FIG. 15C is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at the amino and carboxyl terminal end to rat lung tissue homogenateas a function of absorbance at 650 nm.

FIG. 15D is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at the amino and carboxyl terminal end to rat kidney tissuehomogenate as a function of absorbance at 650 nm.

FIG. 15E is a graph of the binding of SEQ ID NOs: 8 and 14 with a biotintag at the amino and carboxyl terminal end to rat mesenteric lymph nodetissue homogenate as a function of absorbance at 650 nm.

FIG. 16A is a graph of the binding of SEQ ID NO:8 at a concentration of25 μg/ml to intestinal epithelial tissue in the presence of varyingconcentrations of free L-cysteine as a function of absorbance at 650 nm.

FIG. 16B is a graph of the binding of SEQ ID NO:8 at a concentration of12.5 μg/ml to intestinal epithelial tissue in the presence of varyingconcentrations of free L-cysteine as a function of absorbance at 650 nm.

FIG. 16C is a graph of the binding of SEQ ID NO:8 at a concentration of6.25 μg/ml to intestinal epithelial tissue in the presence of varyingconcentrations of free L-cysteine as a function of absorbance at 650 nm.

FIG. 16D is a graph of the binding of SEQ ID NO:25 at a concentration of6.25 μg/ml to intestinal epithelial tissue in the presence of varyingconcentrations of free L-cysteine as a function of absorbance at 650 nm.

FIG. 17A is a graph of the binding of derivatives-of SEQ ID NO:8,including SEQ ID NOs: 31, 32 and 33, to Caco-2 homogenates 1s as afunction of absorbance at 650 nm.

FIG. 17B is a graph of the binding of derivatives of SEQ ID:25,including SEQ ID NOs: 37, 38 and 40, to Caco-2 homogenates as a functionof absorbance at 650 nm.

FIG. 18 is a graph of the binding of SEQ ID NO:3 with a zinc-bindingmotif, SEQ ID NO:43, to Caco-2 homogenates as a function of absorbanceat 650 nm.

FIG. 19A is a graph of the binding of SEQ ID NOs: 25, 31, 32 and 33 thatwere adsorbed to streptavidin particles to Caco-2 cells as a function ofabsorbance at 650 nm.

FIG. 19B is a graph of the binding of SEQ ID NOs: 25 and 40 that wereadsorbed to streptavidin particles to Caco-2 cells as a function ofabsorbance at 650 nm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to targeted polypeptide ligands formucosal delivery of agents through the intestinal epithelium. In oneembodiment of the invention, the polypeptide ligands are targeted toM-cells or Peyer's patch tissue of the intestinal epithelium.

As used herein, the term “purified synthetic polypeptide ligand” isintended to distinguish polypeptides of the invention from (1) thosethat consist of a naturally occurring amino acid sequence; and (2) thosethat naturally occur but have not been purified.

Examples of polypeptides that naturally occur but which have not beenpurified are fragments of polypeptides that exist as intermediatesduring the translational process that elongates fragments into completepolypeptides, and proteolytic breakdown products which occur from timeto time.

The polypeptide component of a protein, such as mouse keratinocytegrowth factor, identified in the Blast homology search below would be anexample of a naturally occurring polypeptide.

A population of synthetic polypeptide ligands in solution, wherein mostor all of the polypeptides in solution are a particular syntheticpolypeptide ligand, is one example of a purified synthetic polypeptideligand.

A polypeptide ligand that may naturally occur in a eukaryotic cell is apurified synthetic polypeptide ligand if it occurs (but does notnaturally occur) on a phage surface or a bacterial surface, or if itoccurs on the surface of a nanoparticle, microparticle or liposome, orbacterial or viral carrier, or if it occurs as a result of geneticrecombination technologies in a cell, virus or phage where it does notnaturally occur.

As used herein, the terms “polypeptide” and “peptide” do not have anintrinsic difference as to biochemical meaning. As indicated herein, a12-mer peptide can qualify as a polypeptide. In a purified syntheticpolypeptide ligand where one or more amino acids have been derivatized(e.g. glycosylation, acetylation, amidation, biotinylation, dansylation)the term purified synthetic polypeptide ligand is intended to apply tothe polypeptide component of the ligand. In the case where dansylationcomprises the addition of a dansyl-lysine group the polypeptide absentthe lysine of the dansyl-lysine group is the purified syntheticpolypeptide ligand.

The test for functionality of a 12-mer, fragment or homologue isexemplified by “wherein said 12-mer L-peptide, fragment or homologuethereof, when integrated as an N-terminal PIII fusion peptide of an M13phage confers an ability to bind the phage to a Caco-2 cell homogenatemembrane fraction, said ability being at least as great as thatconferred by a similarly integrated 12-mer peptide of SEQ ID NO:67.” Acloning vector useful for accomplishing the binding test is M13KE, whichis available from New England Labs Inc., as are the details forintegrating the peptide {See Technical Bulletin #8101(Apr. 1, 2000),which is incorporated by reference herein in its entirety). Peptideslarger than 20-30 amino acids, if so integrated have deleterious effecton the infectivity of the M13 virus. If it is desired to test thebinding functionality of peptides too large to be tested in the phagebinding test, such larger peptides in biotinylated form can be tested inthe Caco-2 membrane binding assay described herein, in order to-see ifthat larger peptide retains detectable binding activity.

The terms “a Caco-2 cell homogenate membrane fraction” and simply“Caco-2 cell homogenate” are used interchangeably herein unlessotherwise indicated. In vivo phage display library screening was used todetermine polypeptide ligands that bind to intestinal Peyer's Patch andnon-Peyer's Patch tissue homogenates of several species. DNA fromone-hundred phage clones with the highest binding affinities (O.D.>0.75)was sequenced to identify the sequence of the peptide insert. Thirtyunique sequences were identified, of which there were several commontripeptide motifs. More than one copy of several clones was isolated andseveral clones were isolated from different rats (See Table 1 below).

The 12-mer peptides and related peptides, a total of 43 in all (SeeTables 1 and 2) were synthesized and used as ligands in binding studies.The related peptides included selected homologues, D-forms andretro-inverted forms of the 12-mers, as well as a zinc-binding chimericpeptide (SEQ ID NO:43).

By employing the foregoing techniques, the inventors have identifiedseveral polypeptide ligands, which mediate binding to intestinalepithelium of several species, including rat, dog, mouse, pig and/orhuman intestinal epithelium tissue. Thus, the invention encompasses thefollowing ligands (Tables 1 & 2): TABLE 1 Amino Acid Sequences forLigands No. of copies of each clone SEQ ID Sequence isolated SEQ ID NO:1ATPPPWLLRTAP 1 SEQ ID NO:2 DGSIHKRNIMPL 1 SEQ ID NO:3 DYDSLSWRSTLH 1 SEQID NO:4 GEPTTDMRWRNP 1 SEQ ID NO:5 GLWPWNPVTVLP 5 SEQ ID NO:6HMLNDPTPPPYW 2 SEQ ID NO:7 KPAYTHEYRWLA 3 SEQ ID NO:8 LETTCASLCYPS 1 SEQID NO:9 LGTDWHSVSYTL 1 SEQ ID NO:10 LGTLNAGVPGFP 1 SEQ ID NO:11LTHSKNPVFLST 1 SEQ ID NO:12 LVPTTHRHWPVT 1 SEQ ID NO:13 LVSNARGFNNLS 1SEQ ID NO:14 NTRIPEPIRFYM 1 SEQ ID NO:15 NVYTFHSMSPMP 1 SEQ ID NO:16QHTTLTSHPRQY 1 SEQ ID NO:17 SDFSDTMPHRPS 2 SEQ ID NO:18 SIDTIQILSLRS 3SEQ ID NO:19 SISWASQPPYSL 1 SEQ ID NO:20 SMVKFPRPLDSR 2 SEQ ID NO:21LRRWVRVWLRL 1 SEQ ID NO:22 TMSPNVYYTAFG 1 SEQ ID NO:23 TQIPSRPQTPSQ 1SEQ ID NO:24 VCSNMYFSCRLS 1 SEQ ID NO:25 VPPHPMTYSCQY 1 SEQ ID NO:26VPRLEATMVPDI 1 SEQ ID NO:27 VPTKPELPVNFT 1 SEQ ID NO:28 WSSDLPQPASTY 1SEQ ID NO:29 VITPYAHLRGGN 5 SEQ ID NO:30 NVYTDNTLSPTP 1

Table 2: Stabilized versions (D-form, retro-D form) and homologues ofthese sequences were also synthesized. (L-form amino acid residues aregiven as capital letters while D-form amino acids are given as-lowercase letters.) SEQ ID Sequence SEQ ID NO:31 LETTAASLCYPS SEQ ID NO:32LETTCASLAYPS SEQ ID NO:33 LETTAASLAYPS SEQ ID NO:34 LETTSASLSYPS SEQ IDNO:35 spyclsacttel SEQ ID NO:36 lettcaslcyps SEQ ID NO:37 vpphpmtyscqySEQ ID NO:38 VPPHPMTYSAQY SEQ ID NO:39 VPPHPMTYSSQY SEQ ID NO:40yqcsytmphppv SEQ ID NO:41 vscnmyfscrls SEQ ID NO:42 VSSNMYFSSRLS SEQ IDNO:43 DYDLSLWRSTLHGGHESSH

Analysis of the 12-mer peptide sequences revealed that several peptidescontain common motifs. Thus, the invention also encompasses these motifsand polypeptide ligands containing the motifs, wherein the polypeptideligands facilitate transport of a pharmaceutical agent into or acrossthe intestinal epithelium, M-cells or Peyer's patch tissue.

The motifs PPY, PVT, LGT and NVY have no previously defined receptor.The motif TPPP has been described as a low affinity omega-opioid peptideantagonist. Certain opioid receptors have been observed on intestinalepithelium. An additional motif of the invention is NVYTXXXXSPXP (SEQ IDNO:98) wherein X is any amino acid.

There are several groups of preferred synthetic polypeptide ligands ofthe invention, wherein the members of each group contain related aminoacid sequences. A first such group contains ligands comprising an aminoacid sequence selected from the group consisting of: LETTCASLCYPS (SEQID NO:8), LETTAASLCYPS (SEQ ID NO:31), LETTCASLAYPS (SEQ ID NO:32),LETTAASLAYPS (SEQ ID NO:33), LETTSASLSYPS (SEQ ID NO:34), spyclsacttel(SEQ ID NO:35) and lettsaslsyps (SEQ ID NO:36). A second such groupcontains ligands selected from the group consisting of: VPPHPMTYSCQY(SEQ ID NO:25), yqcsytmphppv (SEQ ID NO:40), VPPHPMTYSSQY (SEQ ID NO:39)and VPPHPMTYSAQY (SEQ ID NO:38). A third such group contains ligandscomprising an amino acid sequence selected from the group consisting of:VCSNMYFSCRLS (SEQ ID NO:24), vcsnmyfscrls (SEQ ID NO:41) andVSSNMYFSSRLS (SEQ ID NO:42).

Ligands of the invention are useful for transporting a carrier entity orpharmaceutical agent into or across the intestinal epithelium, M-cellsor Peyer's patch tissue. Thus, the invention not only provides novelligands, but also provides a method to transport a carrier entity orpharmaceutical agent into or across the intestinal epithelium, orM-cells or Peyer's patch tissue, as well as novel ligand-entitycomplexes.

As used herein, the term “carrier entity” is defined as a particle,droplet, bacterium, phage or virus that can carry a pharmaceuticalagent. As used herein, the term “carrier entity” is also defined as abacterium, phage or virus that can code for a pharmaceutical agent Amicroparticle is defined as a particle whose “major dimension” is in therange 1 to 5 μm, most preferably in the range 1 to 3 μm. A nanoparticleis defined as a particle whose major dimension is less than 1μ,preferably in the range 1 nm to 500 nm, most preferably in the range 10nm to 500 nm.

As used herein, the major dimension of a spherical particle is itsdiameter, and that of a rod-shaped particle, its length is For otherparticles, it is the longest dimension possible for the particle.

Nano- and microparticles that are loaded with, or encapsulate,pharmaceutical agents, can be coated with the polypeptide ligands, suchas those of the present invention, that target intestinal epitheliumtissue, such as M-cell or Peyer's patch tissue. The coating can beeffected by covalent or non-covalent bonding. The covalent bonding canbe achieved by adsorption or any other coating process. In either case,the bonding can be made to completed particles or to particle componentsthat subsequently form part of the particles.

Biodegradable particles are preferred.

Pharmaceutical agents can, in the alternative, be directly linked topolypeptide ligands. If the agent is itself a polypeptide or peptide,the product is a chimeric polypeptide comprising both an agent and atargeting portion. Bacterial vectors can express a targeting ligand ontheir surface and also express an antigen on their surface or carry agene coding for the antigen. Viral vectors can express a targetingligand on their surface and also express an antigen on their surface orcarry a gene coding for the antigen.

A “pharmaceutical agent” is a therapeutic or diagnostic agent.Therapeutic agents are those that are administered either to treat anexisting disease or prophylactically to protect against a potentialfuture disease. Diagnostic agents are any agents that are administeredas part of a diagnostic procedure.

Examples of therapeutic agents are drugs, genes, gene-delivery vectors,DNA vaccines, antigens and recombinant viruses.

Drugs include, for example, analgesics, anti-migraine agents,anti-coagulant agents, anti-emetic agents, cardiovascular agents,anti-hypertensive agents, narcotic antagonists, chelating agents,anti-anginal agents, chemotherapy agents, sedatives, anti-neoplastics,prostaglandins and antidiuretic agents, antisense oligonucleotides,gene-correcting hybrid oligonucleotides, ribozymes, RNA interference(RNAI) oligonucleotides, silencing RNA (siRNA) oligonucleotides,aptameric oligonucleotides and triple-helix forming oligonucleotides.

Examples of gene-delivery vectors are DNA molecules, viral vectors (E.g.adenovirus, adeno-associated virus, retroviruses, herpes simplex virus,and sindbus virus), and cationic lipid-coated DNA and DNA-dendrimers.

Examples of drugs are as insulin, calcitonin, calcitonin gene regulatingprotein, atrial natriuretic protein, colony stimulating factor,betaseron, erythropoietin (EPO), interferons (E.g. α, β or γinterferon), somatropin, somatotropin, somatostatin, insulin-like growthfactor (somatomedins), luteinizing hormone releasing hormone (LHRH),tissue plasminogen activator (TPA), growth hormone releasing hormone(GHRH), oxytocin, estradiol, growth hormones, leuprolide acetate, factorVIII and interleukins (E.g. interleukin-2). Representative drugs alsoinclude: analgesics (E.g. fentanyl, sufentanil, butorphanol,buprenorphine, levorphanol, morphine, hydromorphone, hydrocodone,oxymorphone, methadone, lidocaine, bupivacaine, diclofenac, naproxen andpaverin); anti-migraine agents (E.g. sumatriptan and ergot alkaloids);anti-coagulant agents (E.g. heparin and hirudin); anti-emetic agents(E.g. scopolamine, ondansetron, domperidone and metoclopramide);cardiovascular agents, anti-hypertensive agents and vasodilators (E.g.diltizem, clonidine, nifedipine, verapamil, isosorbide-5-mononitrate,organic nitrates and agents used in treatment of heart disorders);sedatives (E.g. benzodiazepines and phenothiozines); narcoticantagonists (E.g. naltrexone and naloxone); chelating agents (E.g.deferoxamine); anti-diuretic agents (E.g. desmopressin and vasopressin);anti-anginal agents (E.g. nitroglycerine); anti-neoplastics (E.g.5-fluorouracil and bleomycin); prostaglandins; and chemotherapy agents(E.g. vincristine).

Examples of antigens that are therapeutic agents are tumor antigens,pathogen antigens and allergen antigens. A vaccine preparation willcontain at least one antigen. “Pathogen antigens” are thosecharacteristic of pathogens, such as antigens derived from viruses,bacteria, parasites or fungi.

Examples of important pathogens include vibrio choleras, enterotoxigenicE. Coli, rotavirus, Clostridium difficile, Shigella species, Salmonellatyphi, parainfluenza virus, influenza virus, Streptococcus mutans,Plasmodium falciparum, Staphylococcus aureus, rabies virus andEpstein-Barr virus.

Viruses in general include the following families: picronaviridae;caliciviridae, togaviridae; flaviviridae; coronaviridae; rhabodviridae;filoviridae; paramyxoviridae; orthomyxoviridae; bunyaviridae;arenaviridae; reoviridae; retroviridae; hepadnaviridae; parvoviridae;papovaviridae; adenoviridae; herpesviridae and poxyviridae.

These viruses, especially attenuated versions or otherwise modifiedversions that are not pathogenic, can also be modified to expresstargeting ligands on their surface and thus allow for enhancedvaccination.

Bacteria in general include but are not limited to: P. aeruginosa; E.coli; Klebsiella sp.; Serratia sp; Pseudomanas sp.; P. cepacia;Acinetobacter sp.; S. epidermis; E. faecalis; S. pneumonias; S. aureus;Haemophilus sp.; Neisseria sp.; N. meningitidis; Bacterodies sp.;Citrobacter sp.; Branhamella sp.; Salmonelia sp.; Shigella sp.; S.Lesteria sp., Pasteurella multocida; Streptobacillus sp.; S. pyogenes;Proteus sp.; Clostridium sp.; Erysipelothrix sp.; Spirillum sp.;Fusospirocheta sp.; Treponema pallidum; Borrelia sp.; Actinomycetes;Mycoplasma sp.; Chlamydia sp.; Rickettsia sp., Spirchaeta; Legionellasp.; Mycobacteria sp.; Urealplasma sp.; Streptomyces sp.; Trichomorassp.; and P. mirabilis.

Parasites include but are not limited to: Plasmodium falciparum, P.vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana, L.tropica, L.major, L. aethiopica, L. donovani, Trypanosoma cruzi, T.brucei, Schistosoma mansoni, S. haematobium, S. japonium; Trichinellaspiralis; Wuchereria bancrofti; Brugia malayli; Entamoeba histolytica;Enterobus vermiculoarus; Taenia solium, T. saginata, Trichomonasvaginitis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidiumparvum; Pneumocvtis carinii, Babesia bovis, B. divergens, B. microti,Isospore belli, L. hominis; Dientamoeba fragiles; Onchocerca volvulus;Ascaris lumbricoides; Necator americanis; Ancylostoma duodenale;Strongyloides stercoralis; Capillaria philippinensis; Angiostrongylyscantonensis; Hymenolepis nan; Diphyllobothrium latum; Echinococcusgranulosus, E. multilocularis; Paragonimus westermani, P. caliensis;Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciolahepatica, Sarcoptes scabiei, Pediculus humanus; Phtirius pubis; andDermatobia hominis.

Fungi in general include but are not limited to: Crytpococcusneoformans; Blastomyces dematitidis; Aiellomyces dermatitidisHistoplasfrai capsulatum; Coccidiodes immitis; Candids species,including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondiiand C. krusei, Aspergillus species, including A. fumigatus, A. flavusand A. niger, Rhizopus species; Rhizomucor species; Cunnighammellaspecies; Apophysomyces species, including A. saksenaea, A. mucor and A.absidia; Sporothrix schenckii, Paracoccidioides brasiliensis;Pseudallescheria boydii, Torulopsis glabrata; and Dermatophyres species.

Antigens that are allergens can be haptens, or antigens derived frompollens, dust, molds, spores, dander, insects and foods. Specificexamples include the urusiols of Toxicodendron species and thesesquiterpenoid lactones.

Adjuvants can, if desired, be delivered by the carrier entity or with acarrier entity. Examples of adjuvants are Freund's Complete Adjuvant,Freund's Incomplete Adjuvant, Hunter's Titermax, Gerbu Adjuvant, Ribi'sAdjuvant, Montanide ISA Adjuvant, Aluminum Salt Adjuvants andNitrocellulose adsorbed protein.

Diagnostic agents include antibodies, nucleic acids and imaging agents,as well as molecules that are needed to make such antibodies, nucleicacids or imaging agents detectable.

A preferred method of the invention for administering a carrier entityto an organism having intestinal epithelium comprises contacting theintestinal epithelium with a polypeptide ligand of the invention in thepresence of the carrier entity, such that the carrier entity istransported into or across the intestinal epithelium or into or across apreferred region of the intestine such as M-cells or Peyer's patches.

The carrier entity and the polypeptide ligand can be administeredtogether (E.g., as part of an entity-ligand complex or discretely) orseparately. Oral administration is most preferred, but other modes ofadministration requiring transepithelial transport to reach the targettissue are also acceptable (E.g., rectal administration).

Of course, the ability of the ligands of the invention to target certaincells of the intestinal epithelium also makes the ligands suitable fortargeting pharmaceutical agents to the cells themselves for therapy orprophylaxis.

In addition to the aforementioned ligands and methods, the inventionalso encompasses nucleic acid sequences encoding the ligands of theinvention. As stated herein, the term “encodes” includes the actualcoding sequence or a complementary strand. Preferred nucleic acidsequences of the invention are shown in the following table: TABLE 3Nucleic Acid Sequences of the Targeting Ligands SEQ ID NO:44ATACTGCCTAGGATGAGAAGTCAACGTAGTATGCTG SEQ ID NO:45ATTAGTCTAAGCCACACTCGCACCCAACGTCGGAGA SEQ ID NO:46CCTCGAATCAAGCGGACGAGGAAACTTCACCATAGA SEQ ID NO:47AGGATTCCGCCACCTCATATCCGTAGTCGGCTCACC SEQ ID NO:48AAGCGGCATAATATTCCGCTTATGAATCGAACCATC SEQ ID NO:49ATGAAGAGTAGAACGCCAAGAAAGCGAATCATAATC SEQ ID NO:50ATACGTCGAAGCCGGCTGCGGCAGATCAGACGACCA SEQ ID NO:51AGTAGAAAGAAACACAGGATTCTTAGAATGCGTAAG SEQ ID NO:52AGACAGACGACAAGAAAAATACATATTCGAACAAAC SEQ ID NO:53CTGAGAAGGAGTCTGCGGCCTAGACGGAATCTGAGT SEQ ID NO:54ATTCCCCCCACGCAAATGAGCATAAGGAGTAATATA SEQ ID NO:55CATATAAAACCTAATCGGCTCAGGAATCCTCGTATT SEQ ID NO:56AGAACGAAGCGAAAGAATCTAAATCGTATCAATACT SEQ ID NO:57AGCAAGCCAACGATACTCATGCGTATACGCCGGCTT SEQ ID NO:58CGGCATAGGAGACATAGAATGAAACGTATACACATT SEQ ID NO:59CGTAAAATTAACCGGAAGCTCCGGCTTAGTCGGCAC SEQ ID NO:60AGGAAGAACCGTAACAGGATTCCAAGGCCACAGACC SEQ ID NO:61CCAATAAGGAGGAGGCGTAGGATCATTCAGCATATG SEQ ID NO:62CGACGGATAACACAAACTAGCACAAGTCGTCTCAAG SEQ ID NO:63CGACAGATTATTAAACCCACGAGCATTAGAAACAAG SEQ ID NO:64CGAAGGCCGATGAGGCATAGTATCCGAAAAATCAGA SEQ ID NO:65CATATCAGCAAGAATACGTCATAGGATGAGGAGGCAC SEQ ID NO:66CGGCGTAGGAGACAGAGTATTATCCGTATACACATT SEQ ID NO:67AGGCGCAGTCCGCAGAAGCCAAGGCGGAGGAGTAGC SEQ ID NO:68AAGAGTATACGACACAGAATGCCAATCCGTCCCCAA SEQ ID NO:69AAGAGTATACGACACAGAATGCCAATCCGTCCCCAA SEQ ID NO:70AATATCCGGAACCATCGTCGCCTCAAGACGAGGCAC SEQ ID NO:71CGGAAAACCCGGCACACCAGCATTCAACGTCCCAAG SEQ ID NO:72CGTCACAGGCCAATGACGATGAGTCGTCGGCACAAG SEQ ID NO:73CAAAGAATAAGGAGGCTGCGACGCCCAAGAAATAGA

In addition, arising from the degeneracy of the genetic code allvariations of these DNA sequences resulting in identical amino acidsequences are included in this invention.

The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

The Screening Process: Biopanning In Vivo

A Phage Display Peptide Library (12-mer at 1.5×10¹¹ pfu) was inoculatedintraduodenally into a rat loop model (n=5) Blood samples were takenfrom the rat loop at time periods of 0, 30, 60, 90 and 120 minutes. Theanimals were then sacrificed and the loops excised. From these loops,Peyer's patch and non-Peyer's patch tissues were isolated, washed andhomogenized. The bacteriophage present in the tissue samples wereamplified in E. Coli and isolated by polyethylene glycol (PEG)precipitation. Peyer's patch specific phage were titred and selected foruse in subsequent screening cycles.

Four screening cycles were completed and phage titres (in pfu/ml) wereobtained at cycle 4. TABLE 4 Phage titres (pfu/ml) in crude tissuehomogenates (rat loop model; cycle 4) Rat No. Peyer's Patch 1 1.0 × 10⁶2 5.6 × 10⁵ 3 4.0 × 10³ 4 2.6 × 10⁶ 5 4.6 × 10⁴

Example 2

Phage Binding Studies

The phage pools obtained in Example 1 were plated out on LB agar plateswith top agar and phage clones were selected for evaluation by an ELISAanalysis of binding to Peyer's patch tissue from various species, alongwith Caco-2 and IEC-6 cell models.

The ELISA was run with 5 μg/ml of phage homogenates, Blocking buffer: 1%BSA-TBS, wash buffer:TBS-Tween (0.05%), anti-M13 biotin conjugate(Research Diagnostics RDI-PRO61597) at a 1:5000 dilution, ExtrAvidinAlkaline Phosphatase (Sigma E-2636) at a 1:5000 dilution and pNPPsubstrate.

Five hundred clones (Rats 1-5 from Cycle 4) were subsequently assayedusing the above method. (See FIGS. 1A-5 for binding profiles). Theclones exhibited a broad range of activity with concentration-dependentbinding clearly detectable for all high-binding clones. TABLE 5 Table ofphage clone numbers & SEQ ID NOs. SEQ ID of No. of copies ofcorresponding SEQ ID NO: Amino Acid Sequence Clone No. DNA sequence SEQID NO:1 ATPPPWLLRTAP 1 3.030 SEQ ID NO:67 SEQ ID NO:2 DGSIHKRNIMPL 11.010 SEQ ID NO:48 SEQ ID NO:3 DYDSLSWRSTLH 1 1.016 SEQ ID NO:49 SEQ IDNO:4 GEPTTDMRWRNP 1 1.008 SEQ ID NO:47 SEQ ID NO:5 GLWPWNPVTVLP 5 2.054,3.003, SEQ ID NO:60 3.056, 5.006, 5.074 SEQ ID NO:6 HMLNDPTPPPYW 22.061, 4.075 SEQ ID NO:61 SEQ ID NO:7 KPAYTHEYRWLA 3 2.025, 2.068, SEQID NO:57 3.083 SEQ ID NO:8 LETTCASLCYPS 1 2.078 SEQ ID NO:62 SEQ ID NO:9LGTDWHSVSYTL 1 4.009 SEQ ID NO:69 SEQ ID NO:10 LGTLNAGVPGFP 1 5.039 SEQID NO:71 SEQ ID NO:11 LTHSKNPVFLST 1 1.049 SEQ ID NO:51 SEQ ID NO:12LVPTTHRHWPVT 1 5.049 SEQ ID NO:72 SEQ ID NO:13 LVSNARGFNNLS 1 2.081 SEQID NO:63 SEQ ID NO:14 NTRIPEPIRFYM 1 2.014 SEQ ID NO:55 SEQ ID NO:15NVYTFHSMSPMP 1 2.045 SEQ ID NO:58 SEQ ID NO:16 QHTTLTSHPRQY 1 1.002 SEQID NO:44 SEQ ID NO:17 SDFSDTMPHRPS 2 3.006, 3.090 SEQ ID NO:64 SEQ IDNO:18 SIDTIQULSLRS 3 2.016, 3.014, SEQ ID NO:56 3.031 SEQ ID NO:19SISWASQPPYSL 1 5.078 SEQ ID NO:73 SEQ ID NO:20 SMVKFPRPLDSR 2 1.005,1.076 SEQ ID NO:46 SEQ ID NO:21 LRRWVRVWLRL 1 1.004 SEQ ID NO:45 SEQ IDNO:22 TMSPMVYYTAFG 1 3.062 SEQ ID NO:68 SEQ ID NO:23 TQIPSRPQTPSQ 11.099 SEQ ID NO:53 SEQ ID NO:24 VCSNMYFSCRLS 1 1.083 SEQ ID NO:52 SEQ IDNO:25 VPPHPMTYSCQY 1 3.020 SEQ ID NO:65 SEQ ID NO:26 VPRLEATMVPDI 14.098 SEQ ID NO:70 SEQ ID NO:27 VPTKPELPVNFT 1 2.049 SEQ ID NO:59 SEQ IDNO:28 WSSDLPQPASTY 1 1.038 SEQ ID NO:50 SEQ ID NO:29 YITPYAHLRGGN 52.012, 3.005, SEQ ID NO:54 3.013, 3.035, 5.033 SEQ ID NO:30 NVYTDNTLSPTP1 1.009 SEQ ID NO:66

Example 3

Specificity Determination: Analysis of Phage Binding to Rat SmallIntestine and/or Peyer's Patch

A Biotin-ExtrAvidin Alkaline Phosphatase assay was established for highthroughput screening of the phage clones. The initial screens identified55 out of the 500 clones as high-binding clones (an absorbency readingof >0.75).

The rat tissue homogenates were prepared by harvesting rat GI andPeyer's patch and storing them on ice until needed, or 1-2 hours. Thetissue was then put into homogenization buffer (250 mM Sucrose, 12 mMTris, 16 mM EDTA) with protease inhibitor cocktail. A hand-heldhomogenizer was used to break up the tissue for 3-4 minutes. Thecontents of the homogenizer were then transferred to microfuge tubes andspun at 1500 rpm for 1 minute. The supernatant was taken off andmeasured for protein content using the Bio-Rad Assay. Specificitystudies were then run to allow differentiation between Peyer's patchspecific and non-specific binding properties. The 55 high-binding cloneswere assayed for binding to rat small intestinal homogenates (i.e.,homogenate membrane fractions) with and without Peyer's Patch tissue(i.e., tissue homogenate membrane fractions) present. (See FIGS. 6 and7). The negative control was M13mp18 with no peptide insert. Thisnegative control consistently showed absorbance readings of <0.200. Allof the clones exhibited significantly higher binding to both tissuetypes as compared to the control. However, there was a negligibledifference between binding to Peyer's patch and non-Peyer's Patchtissue, which suggests that the clones are binding to factors common toboth tissue types.

These results were reproducible when using a further 50 clones with anabsorbance reading between 0.5-0.75.

2. Species Specificity: Analysis of Phage Binding to Pig, Dog and MouseSmall Intestine and/or Peyer's Patch

One hundred high-binding clones were assayed for their bindingproperties to pig, dog and mouse small intestinal homogenates that werewith and without Peyer's patch tissue homogenates. (See FIGS. 8, 9 and10). These homogenates were prepared in the same way as the method forobtaining rat homogenates described above. As was observed with the rattissues, all the clones exhibited a negligible difference betweenbinding to Peyer's patch and non-Peyer's patch tissue suggesting thathuman colon epithelial adenocarcinoma cell line believed to displayproperties of human small intestinal epithelial cells.

In order to prepare the Caco-2 cell membrane and cytosolic fractions,confluent Caco-2 cell monolayers (grown in 75 cm² flasks for up to 1week at 37° C. and 5% CO₂) were washed twice in Dulbecco's PBS (DPBS).The cell monolayers were then treated with 10 mM EDTA-DPBS for 5-10minutes at 37° C. and the cells were harvested by centrifugation at 1000rpm for 5 minutes. The cells were then washed 3× in DPBS. The cellpellet was resuspended in 3 volumes of ice-cold HED buffer (20 mM HEPES(pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsuphonylfluoride (PMSF)) and the cells were allowed to swell for 5 minutes onice. The cells were then homogenized for 30 seconds. The cellhomogenates were then centrifuged in hard walled tubes at 40,000 rpm for45 minutes at 4° C. (ultracentrifuge Ti90 rotor) The supernatant wasremoved and the pellet resuspended in HEDG buffer (20 mM HEPES (pH7.67), 1 mM EGTA, 0,5 mM dithiothreithol, 100 mM NaCl, 10% glycerol, 1mM PMSF). Three volumes of buffer. were then added and the pellet wasresuspended and centrifuged again at 1000 rpm for 2 minutes. Thesupernatant was removed and stored on ice. The procedure was repeatedadding the second supernatant to the first and then the procedure wasrepeated 2-3 more times. The protein concentration was determined usingthe Bio-Rad protein assay. All fractions were stored at −80° C.

The IE-6 cell homogenates were prepared in the same way as the Caco-2homogenates as described above.

Analysis of binding of phage clones to Caco-2 cell membrane fractions byELISA was done as follows: 96-well ELISA plates were coated overnight at4° C. with Caco-2 cell membrane fractions (10 μg/ml in 0.05M carbonatebuffer (pH9.6); 100 μl/well). The plates were then blocked in 1.5%BSA-TBS for 1 hour at room temperature (100 μl/well) prior to washing 3×in TBS/Tween 20 (0.05%). Phage clones (1:2 dilution in 1.5% BSA-TBS)were serially diluted down the plate and incubated for one-two hours atroom temperature. After 3 washes in TBS/Tween 20 the phage wereincubated with biotinylated mouse anti-M13 MAb (1:5000 dilution in 1.5%BSA-TBS; RDI; 100 μl/well) for one hour at room temperature. The plateswere washed three times prior to incubation with extravidin AP (1:5000dilution in 1.5% BSA-TBS; Sigma; 100 μl/well) at room temperature forone hour. The plates were again washed 3 times in TBS/Tween 20. Alkalinephosphatase activity was detected using the substrate p-NPP(p-nitrophenyl phosphate). After 30 minutes, development of theenzymatic reaction was stopped by addition of 3M NaOH (100 μl/well). Theplates were read at 405 nm using an ELISA plate reader.

The clones showed a broad range of activity with high binders exhibitingconcentration-dependent binding. The binding profiles showednon-differentiated and differentiated Caco-2 cell fractions givingsimilar results. The absorbance readings varied between the differenttissues and cell types, however, the overall binding profile remainedunchanged. (See FIGS. 11 and 12).

Example 4

Sequencing of Selected Phage Clone Inserts

The 100 phage clones from Example 3 including all high-binding clonesand a selection of medium- and low-binding clones were sequenced todetermine the nature of the peptide inserts.

The phage DNA was isolated using Qiagen's Quiaprep M13 spin kits. Theisolated DNA was precipitated and subsequently sequenced with a 96 gIIIsequencing primer situated 117 base pairs 3′ of the peptide insert.

Of the 100 inserts sequenced, 53% did not contain a detectable insert ingene III and all of these clones represented the low binding clones withan absorbance of <0.4. This appeared to correspond with the libraryphage (MP13KE) and may have represented loss of the insert during finalamplification of selected clones or may have been selected by theirability to be taken up as particulate matter in either Peyer's patchM-cells or enterocytes or alternatively these clones may have a mutationelsewhere in gene III, gene VII or another gene of M13 which wasselected during the screening program for binding to/uptake intointestinal epithelium or Peyer's patch tissue in vivo.

A BLAST search using the Swissprot database was performed on thirty ofthe unique sequences in order to compare the predicted peptide sequenceto the protein/peptide sequence database. A summary of BLAST alignmentsis as follows: TABLE 6 Alignment of most relevant Blast homologues. SEQID Sequence Homologue of interest Alignment (SEQ ID NO:74) SEQ ID NO:4GEPTTDMRWRNP MOUSE KERATINOCYTE Query: 1 GEPTTDMRW 9 GROWTH FACTOR GPT+ MRW RECEPTOR Sbjct: 183 GNPTSTMRW 191 (SEQ ID NO:75) SEQ ID NO:5GLWPWNPVTVLP HUMAN UROKINASE-TYPE Query: 4 PWNPVTVL 11 PLASMINOGENPWN  TVL ACTIVATOR PRECURSOR Sbjct: 93 PWNSATVL 100 (u-PA) (SEQ IDNO:76) SEQ ID NO:6) HMLNDPTPPPY XENLA EPITHELIAL- Query: 4 NDPTPPPY 11CADHERIN PRECURSOR NDPT PPY (E-CADHERIN) Sbjct: 811 NDPTAPPY 818 (SEQ IDNO:77) SEQ ID NO:7 KPAYTHEYRWLA PRECURSOR (FCεRI) Query: 4 YTHEYRWL 11(IGE FC RECEPTOR, YT EYRWL ALPHA-SUBUNIT) Sbjct: 196 YTIEYRWL 203(FC-EPSILON-RI- ALPHA) (SEQ ID NO:78) SEQ ID NO:8 LETTCASLCYPS CLALULECTIN-RELATED Query: 2 ETTCASLYCPS 12 PROTEIN PRECURSOR ET  ASL YPS(CLLRP) (LRPCL) Sbjct: 210 ETLIASLTYPS 220 (SEQ ID NO:79) CAVPO CASEIN AQuery: 2 ETTCASLC 9 PRECURSOR ET CASLC 9 Sbjct: 48 ETICASLC 55 (SEQ IDNO:80) SEQ ID NO:9 LGTDWHSVSYTL PIG ZONADHESIN Query: 1 LGTDWHSVSYT 11PRECURSOR LGTDW S + T Sbjct: 750 LGTDWFSPNCT 760 (SEQ ID NO:81) SEQ IDNO:10 LGTLNAGVPGFP MOUSE ELASTIN Query: 4 LNAGVPGF 11 PRECURSOR L AGVPGF(TROPOELASTIN) Sbjct: 612 LGAGVPGF 619 (SEQ ID NO:82) Query: 6 AGVPGF 11AGVPGF Sbjct: 623 AGVPGF 628 (SEQ ID NO:83) Query: 6 AGVPGF 11 AGVPGFSbjct: 632 AGVPGF 637 (SEQ ID NO:84) Query: 6 AGVPGF 11 AGVPGF Sbjct:641 AGVPGF 646 (SEQ ID NO:85) Query: 6 AGVPGF 11 AGVPGF Sbjct: 650AGVPGF 655 (SEQ ID NO:86) (SEQ ID NO:12 LVPTTHRHWPVT MOUSE STROMELYSIN-3Query: 3 PTTHRHWPV 11 PRECURSOR (MATRIX P +HRH PV METALLOPROTEINASE-Sbjct: 40 PESHRHHPV 48 11) (MMP-11) (SEQ ID NO:87) SEQ ID NO:15NVYTFHSMSPMP RAT SUCRASE- Query: 1 NVYTFHSMSPMP 12 ISOMALTASE, NYT  S+ P+P INTESTINAL Sbjct: 987 NPYTLTSIQPLP 99 (SEQ ID NO:88) SEQ IDNO:16 QHTTLTSHPRQY HUMAN PLACENTAL- Query 2: HTTLTSHP 9 CADHERINPRECURSOR H T+T+HP (P-CADHERIN) Sbjct: 376 HFTITTHP 383 (SEQ ID NO:89)SEQ ID NO:19 SISWASQPPYSL CAPHI BETA CASEIN Query: 3 SWASQPPYSL 12PRECURSOR SW  QPP  L Sbjct: 157 SWMHQPPQPL 166 (SEQ ID NO:90) SEQ IDNO:20 SMVKFPRPLDSR ZO1 MOUSE TIGHT Query: 6 PRPLDSR 12 JUNCTION PROTEINZO1 PR LDSR (TIGHT JUNCTION Sbjct: 1110 PRDLDSR PROTEIN 1) SEQ ID NO:91)SEQ ID NO:23 TQIPSRPQTPSQ MOUSE VERSICAN CORE Query: 1 TQIPSRPQTPS 11PROTEIN PRESURSOR T++P  P TPS (LARGE FIBROBLAST Sbjct: 1173 TELPKFPSTPS1183 PROTEOGLYCAN) (SEQ ID NO:92) SULFATE PROTEOGLYCAN Query: 1TQIPSRPQTPS 11 CORE PROTEIN 2) (PGM) T IPS PQ P+ Sbjct: 307 TGIPSTPQKPT317 (SEQ ID NO:93) SEQ ID NO:25 VPPHPMTYSCQY PAPCY Query: 1 VPPHMTYSC 10METALLOPROTEINASE VPPHP T  C INHIBITOR Sbjct: 27 VPPHPQTAFC 36 PRECURSOR(TIMP-1) (SEQ ID NO:94) SEQ ID NO:26 VPRLEATMVPDI HUMAN VERSICAN COREQuery: 1 VPRLEATMVPDI 12 PROTEIN PRECURSOR +PR  AT++P+I (LARGEFIBROBLAST Sbjct: 2695 IPRKSATVIPEI PROTEOGLYCAN) (CHONDROITIN SULFATEPROTEOGLYCAN SULFATE PROTEOGLYCAN CORE PROTEIN 2) (GLIALHYALURONATE-BINDING PROTEIN) (GHAP) (SEQ ID NO:95) SEQ ID NO:27VPTKPELPVNFT HUMAN COLLAGEN ALPHA Query: 1 VPTKPELPVN 10 1 (VII) CHAINVPT PELPV+ PRECURSOR (LONG- Sbjct: 500 VPTGPELPVS 509 CHAIN COLLAGEN)(SEQ ID NO:96) SEQ ID NO:29) YITPYAHLRGNN RAT INSULIN-LIKE Query: 4PYAHLRGG 11 GROWTH FACTOR I PYAH+ GG RECEPTOR Sbjct: 1348 PYAHMNGG 1355

The homologue for SEQ ID NO:4 was found to be keratinocyte growth factorreceptor (KGFR) which is expressed by intestinal as well as otherepithelial cells. It interacts with KGF, a member of the fibroblastgrowth factor (FGF) family of mitogens which is produced by stromalcells and results in epithelial cell proliferation. The KGF-KGFRinteraction is thought to play a role in the epithelial repairprocesses. See Werner, Cytokine Growth Factor Rev, 2:153-65, 1998;Bajaj-Elliott M. et al., J Clin Invest 102:1473-80, 1998.

The homologue for SEQ ID NO:5 is urokinase plasminogen activator (u-PA)which is one of the mediators of the plasminogen activator system, thatalso includes tissue-type plasminogen activator (t-PA) and plasminogenactivator inhibitor type-1 (PAI-1). u-PA cleaves plasminogen to theactive plasmin which can degrade components of the extracellular matrix(ECM). The u-PA receptor (u-PAR) has been shown to be expressed ondifferent types of epithelial cells including intestinal epithelium. SeeGibson P. et al., Gut 7:969-75, 1994. Targeting of the u-PAR has alsobeen shown to enhance gene delivery. See Drapkin P. T. et al., J ClinInvest 105:589-96, 2000.

SEQ ID NO:6 was found to be homologous to cadherin precursors. Cadherinsare epithelial adhesion molecules which allow an intact, selectivelypermeable, epithelial layer to be formed. They are transmembraneglycoproteins that form a complex with cytoplasmic proteins, termedcatenins because they link cadherin to the actin cytoskeleton. TheE-cadherin/catenin interaction is important in intestinal epithelialcells and tight junction integrity. See Jawhari A. et al., Gut 5:581-4,1997.

The homologue of SEQ ID NO:7 is a high affinity FcεRI alpha subunitwhich is a type I transmembrane protein that binds to the Fc region ofIgE. In humans, FcεRI plays a role in the activation of mast cells andbasophils, and participates in IgE-mediated antigen presentation. FcεRIis therefore central to the induction and maintenance of an allergicresponse and may confer physiological protection in parasiticinfections. See Turner H. and Kinet J. P., Nature 402:B24-30, 1999. Thisprotein is expressed on mast cells, eosinophils, Langerhans cells,dendritic cells and monocytes.

SEQ ID NO: 8 shows a strong homology with a lectin-related proteinhowever, unlike true lectins previously shown to bind glycocalyx onenterocytes, this protein is devoid of carbohydrate binding activity.See Van Damme E. J. et al., Plant Mol Biol 3:579-98, 1995.

The homologue of SEQ ID NO: 9 is a zonadhesion precursor which promotesadhesion of spermatozoa to egg extracellular matrix. Hardy D. M. et al.,J Biol Chem. 44:26025-8, 1995. It contains adhesive glycoprotein vonWillebrand's factor domains which share similarity to intestinal mucin,muc2. It is within one of these domains that homology to the selectedpeptide is observed.

The homologue of SEQ ID NO:10 is elastin, a major structural protein ofextracellular matrix. In mouse, the matrix metalloproteinase matrilysin,for which elastin is a substrate, is found in epithelial cells of theuterus, small intestine and extra-testicular ducts. See Wilson C. L. andMatrisian L. M., Int J Biochem Cell Biol 2:123-36, 1996. Interestingly,the 6-residue peptide motif that shares homology with mouse elastin isrepeated five times within the protein while a 4-residue motif withinthis sequence is repeated thirteen times.

SEQ ID NO:12 shows homology to stromelysin, or MMP-3, which isresponsible for the breakdown of ECM collagen as well as the cleavage ofu-PA. See Ugwu F. et al., Biochemistry 2:7231-6, 1998. It plays animportant role together with other members of the MMP family inintestinal tissue remodeling and repair. See Pender S. L. et al., Ann NYAcad Sci 878:581-2, 1999. Peptide SEQ ID NO:25 showed homology to TIMP-1(tissue inhibitor of MMPs). This protein forms irreversible complexeswith MMPs thus inactivating them. It is present in the intestine where,together with MMPs, it is important in the ongoing repair and renewalthat takes place in the intestine.

The homologue of SEQ ID NO:15 was intestinal sucrase-isomaltase which isa brush border hydrolase expressed in epithelial cells located on villi.The greatest amount of the hydrolase is located at the crypt-villusjunction and in the lower to mid-villus region. See Traber P. G.,Biochem Biophys Res Commun 173:765-73, 1990. It has been shown to bedown regulated on M-cells in an in vitro co-culture model. See KerneisS. et al., Science 277:949-52, 1997.

Two different peptides, SEQ ID NO:8 and SEQ ID NO:19, show homology withcasein A and casein B, respectively. Casein is a milk protein and hasbeen shown to bind to small intestinal brush border membranes. See BolteG. et al., J Biochem Biophys Methods 34:189-203, 1997.

One of the selected peptides, SEQ ID NO:20, shares homology with mousetight junction protein, ZO-1. See Itoh M. et al., J Cell Biol 3:491-502,1993. The N-terminus may be involved in transducing a signal requiredfor tight junction assembly, while the C-terminus may have specificproperties of tight junctions. ZO-1 has been shown in vitro to interactwith cadherins.

Several peptides, SEQ ID NOS:23, 26 and 27, show regions of homologywith ECM proteins including versican and collagen. These proteins andproteoglycans are important in tissue integrity acting not only as theglue connecting cells together but also acting in cell motility, growthand differentiation.

SEQ ID NO:29 shows homology to high affinity insulin-like growth factorI receptor (IGF IR) from several species. IGF IR has wide tissueexpression including epithelial cells of the intestine where it acts asan epithelial cell growth factor. See Wolpert S. I. et al., J Surg Res63:345-8, 1996. IGF is a candidate for total parenteral nutrition.

Example 5

Peptide sequences (SEQ ID NOs: 3, 4, 5, 7, 8, 9, 10, 11, 14, 15, 16, 17,19, 20, 26, 28, 29, 30) were synthesized with biotin tags at the aminoterminal for all peptides and additionally at the carboxyl terminal forSEQ ID NOs:8 and 14. Binding of these peptides to Caco-2 homogenatesand/or to rat intestinal tissue homogenates was tested in an ELISA-basedassay with streptavidin-peroxidase detection. High binding to bothtissue types was observed with the following peptides: SEQ ID NOs:3, 8,25 and 24. (See FIGS. 13A-13N).

Example 6

Peptide sequences (SEQ ID NOs:3, 7, 8, 9 and 14) were synthesized withbiotin tags at the amino terminal for all peptides and additionally atthe carboxyl terminal for SEQ ID NOs:8 and 14. Binding of these peptidesto intestinal tissue of different species namely dog, mouse, pig, andrat was performed. No major differences in binding profiles to tissue ofdifferent species were observed. (See FIGS. 14A-14H). Binding ofpeptides (SEQ ID NOs:8 and 14) to rat tissue from different organs,namely liver, lung, mesenteric lymph nodes, spleen and kidney, wasperformed. No major differences in binding profiles to these tissueswere observed. (See FIGS. 15A-15E).

Example 7

Three synthetic peptides (SEQ ID NO:8, 25 and 14) derived from isolatedclones were biotinylated and tested for binding to human Peyer's patchtissue sections. In addition, a known negative binding peptide wasincluded as a negative control. Paraffin sections of human Peyer's patchwere deparaffinized and dehydrated. The sections were rinsed in PBS. Theantigenic determinants on the tissue were unmasked by microwaving in 2.1g/L acetic acid for 5 minutes and allowed to cool at room temperaturefor 20 minutes while covered in plastic wrap. After 20 minutes, thesections were washed in PBS and then blocked in endogenous peroxidase in1% hydrogen peroxide in methanol for 10 minutes. The rinse was repeatedand the sections were blocked with 2% BSA in PBS for 20 minutes at roomtemperature. The sections were incubated with peptide at 50 μg/ml in 2%BSA in PBS for 1 hour at room temperature. Control tissue was treatedwith BSA alone. The sections were rinsed with 0.05% Tween in PBS.Streptavidin-HRP at 1/500 in 2% BSA was added for 30-60 minutes at roomtemperature. Again, the sections were rinsed with PBS/Tween. DABsubstrate was added for up to 5 minutes, and the reaction was stopped byimmersing the slides in water. The sections were counterstained usingHaematoxlin for 50 seconds and then rinsed in water. The slides weredifferentiated in 1% acid alcohol for 5-10 seconds and then rinsed inwater. The slides were mounted using aqueous mounting medium and a coverslip. The negative control peptide showed no binding. SEQ ID NO:14 (alow to medium binder as determined by ELISA) was also negative forbinding in this study. Positive binding to human Peyer's patch wasobserved with SEQ ID NO: 8 and 25. Both peptides gave positive stainingon the apical side of human enterocytes.

Example 8

Cysteine Binding Studies

Peptide SEQ ID NO:8, at a concentration of 6.25 μg/ml, 12.5 μg/ml and 25μg/ml, was co-incubated with L-cysteine over the concentration range of100 mM to 0.003 mM. Peptide SEQ ID NO:25, at a concentration of 6.25μg/ml was co-incubated with L-cysteine over the concentration range of100 mM to 0.003 mM. The presence of free L-cysteine prevented bindingtherefore demonstrating that the cysteine groups are also involved inthe binding of these peptides (See FIGS. 16A-16D).

Discussion of Examples

SEQ ID NO:8 is a medium binder to intestinal epithelial tissue. Wheneither of the two cysteine residues are substituted with an alanineresidue (SEQ ID NOs: 31 and 32), binding is still retained. When bothcysteines are substituted with an alanine residue (SEQ ID NO:33),binding to the epithelium is abrogated. When a biotin tag is added toeither is the amino or carboxyl end, no difference in the bindingaffinities is observed (See FIG. 17A).

SEQ ID NO:25 is the highest binder of the phage-derived peptides. Whenthe cysteine residue is substituted with an alanine residue (SEQ IDNO:38), binding to the epithelium is abrogated. The stabilized D-form(SEQ ID NO:37) and retro-inverted D-form (SEQ ID NO:40) retained highbinding (See FIG. 17B).

SEQ ID NO:24 is a medium binder. When the cysteine residue issubstituted with an alanine residue, the binding to the epithelium isabrogated.

Example 9

A derivative of SEQ ID NO:3 with an added Zinc-binding motif (HESSH) atthe carboxyl terminal (SEQ ID NO:43) was tested for binding to Caco-2homogenates. Enhanced binding was observed with this additional motif(See FIG. 18)

Example 10

Several phage-derived peptides (SEQ ID NOs: 25, 31, 32, 33, and 40) wereadsorbed to streptavidin particles (0.289 μm). Peptide coated particleswere tested in an ELISA-based assay for binding to Caco-2 homogenates.Binding was retained in all the cases that were examined. In addition,when one double-mutant peptide ligand (SEQ ID NO:33), is adsorbed topolystyrene particles binding that is approximately 60% of that of theparent peptide is observed (See FIGS. 19A-19B).

Example 11

Biotinylated SEQ ID NO:40 was adsorbed to the surface of fluorescentpolystyrene particles (0.289 μm) using routine methodologies at roomtemperature. Mouse intestinal loops containing one or more polystyrenesuspensions (typically 300 μl containing 5.0×10¹⁰ particles per ml) wereincubated for 30 minutes. The Peyer's patches were excised, fixed inmethanol and the M cells were counter-stained with UEA1-rhodamine forsubsequent analysis by confocal microscopy. Stained tissues wereexamined on a BioRad MRC 600 confocal laser-scanning microscope.

Fluorescent particles coated with peptide SEQ ID NO:40 exhibited bindingand uptake into M-cells. No binding or uptake was visible using thecontrol streptavidin particles.

Example 12

Titration of Blood Samples for Investigation of Phage Translocation fromGut into Blood

High-binding phage clones numbers 1.009, 5.074, 2.078 and 4.009 wereinjected into rat intestinal loops as described earlier. There were 3mice per group. Blood samples were taken at 0, 30, 60, 90 and 120minutes. The animals were then sacrificed and the loops excised. 100 μlof each blood sample was serially diluted in LB and plated out in topagar plates containing IPTG/Xgal. Blue plaques were counted afterincubation at 37° C. overnight. There was a PBS control and an m13mp19control. TABLE 8 Titration of blood samples for translocation of phagefrom the gut into the blood Phage Corresponding Blood No. of Total phageper Clone No. SEQ ID No. Dilution plaques 100 μl of blood 1.009 SEQ IDNO: 30 Neat 19 19 5.074 SEQ ID NO: 5 Neat 0 0 2.078 SEQ ID NO: 8 10⁻⁶ 454.5 × 10⁷ 4.009 SEQ ID NO: 9 Neat 34 34 M13mp18 — 10⁻¹ 18 180 PBS — Neat1 1 control

Example 13

SEQ ID NO:8 is one of the preferred phage-derived peptides. It is amedium binder. When either of the two cysteine residues are substituted,binding is abrogated. However, when this double-mutant peptide ligand isadsorbed to polystyrene particles binding to approximately 60% of thatof the parent peptide is observed. This may indicate that conformationof the peptides is important. This 12-mer has been synthesized with abiotin tag at either the amino or carboxy end. No differences in bindingaffinities were observed with the addition of the biotin tag. Thestabilized D-form and retro-inverted D form retained high-binding. TABLE9 Results of further studies with SEQ ID NO:8 SEQ ID Binding Assay NO:Comment Sequence results (Caco-2)  8 dansyl-lysine LETTCASLCYPS Highbinder derivative (Kd˜1 μg/ml)  8 biotinylated LETTCASLCYPS Mediumbinder derivative (Kd˜8 μg/ml)  8 LETTAASLCYPS Medium binder 31biotinylated LETTAASLCYPS Medium binder derivative (Kd˜9 μg/ml) 32biotinylated LETTCASLAYPS Medium binder derivative (Kd˜8 μg/ml) 33biotinylated LETTAASLAYPS No binding derivative 35 inverted D-form;spyclsacttel Medium binder biotinylated (Kd˜13.91 μg/ml) derivative 36D-form; lettcaslcyps (Kd˜30.53 μg/ml) biotinylated derivative 34biotinylated LETTSASLSYPS No binding derivative

Example 14

SEQ ID NO:25 is the highest binder of the phage derived peptides. Whenthe cysteine residue is substituted with an alanine residue binding isabrogated. The stabilized D-form and retro-inverted D-form of SEQ IDNO:25 retained high binding. TABLE 10 Results of further studies withSEQ ID NO:25 SEQ ID Binding Assay NO: Comment Sequence results (Caco-2)24 Dansyl-lysine VPPHPMTYSCQY High binder derivative (Kd < 1 μg/ml) 25biotinylated VPPHPMTYSCQY High binder derivative (Kd < 5 μg/ml) 38biotinylated VPPHPMTYSAQY No binding derivative 37 D-form; vpphpmtyscqy(Kd˜10 μg/ml) biotinylated derivative 40 retro D-form yqcsytmphppv(Kd˜10 μg/ml) of SEQ ID NO: 25; biotinylated derivative 39 biotinylatedVPPHPMTYSSQY No binding derivative

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

1. A purified synthetic polypeptide ligand comprising a 12-merL-peptide, fragment or homologue thereof, said 12-mer L-peptide selectedfrom the group consisting of SEQ ID NOs:1-34, SEQ ID NOs:38-39, and SEQID NO:42 wherein said fragment is at least five contiguous amino acidsand wherein said homologue is at least 9/12 homologous to a 12-merpeptide selected from said group and wherein said 12-mer L-peptide,fragment or homologue thereof, when integrated as an N-terminal PIIIfusion peptide of an M13 phage confers an ability to bind the phage toeither Caco-2 cell, IEC-6 cell, rat, mouse, pig or dog homogenatemembrane fractions, said ability being at least as great as thatconferred by a similarly integrated 12-mer peptide of SEQ ID NO:67.
 2. Apurified synthetic polypeptide ligand of claim 1, wherein said ligandcomprises a zinc-binding domain.
 3. A purified synthetic polypeptideligand of claim 1, wherein the homologue is at least 10/12 homologous toa 12-mer peptide.
 4. A purified synthetic polypeptide ligand of claim 1,wherein the homologue is at least 11/12 homologous to a 12-mer peptide.5. A purified synthetic polypeptide ligand of claim 1, wherein saidligand comprises a 12-mer. L-peptide selected from the group consistingof SEQ ID NOs:1-34, SEQ ID NOs:38-39, SEQ ID NO:42.
 6. A purifiedsynthetic polypeptide ligand of claim 1, wherein said ligand consists ofan amino acid sequence selected from the group consisting of SEQ IDNOs:1-34, SEQ ID NOs:38-39, SEQ ID NO:42, fragment thereof or homologuethereof.
 7. A purified nucleic acid sequence encoding for a purifiedsynthetic polypeptide ligand of claim 5, said nucleotide sequence is notmore than 600 nucleotides in length.
 8. A purified synthetic polypeptideligand of claim 1, wherein said ligand consists of a 12-mer L-peptideselected from the group consisting of SEQ ID NOs:1-34, SEQ ID NOs:38-39,SEQ ID NO:42.
 9. A purified synthetic polypeptide ligand of claim 1,wherein said fragment is at least 8 contiguous amino acids.
 10. Apurified synthetic polypeptide ligand of claim 1, wherein said ligand isat most 200 amino acids in length.
 11. A purified synthetic polypeptideligand of claim 1, wherein said ligand is at least 12 amino acids inlength.
 12. A purified synthetic polypeptide ligand of claim 1, whereinsaid polypeptide ligand is at least 30 amino acids in length.
 13. Apurified synthetic polypeptide ligand of claim 1, wherein the 12-merL-peptide is selected from the group consisting of LETTCASLCYPS (SEQ IDNO:8), LETTAASLCYPS (SEQ ID NO:31), LETTCASLAYPS (SEQ ID NO:32),LETTAASLAYPS (SEQ ID NO:34).
 14. A purified synthetic polypeptide ligandof claim 1, wherein the 12-mer L-peptide is LETTCASLCYPS (SEQ ID NO:8).15. A purified synthetic polypeptide ligand of claim 1, wherein the12-mer L-peptide is selected from the group consisting of VPPHPMTYSCQY(SEQ ID NO:25), VPPHPMTYSSQY (SEQ ID NO:39) and VPPHPMTYSAQY (SEQ IDNO:38).
 16. A purified synthetic polypeptide ligand of claim 1, whereinthe 12-mer L-peptide is VPPHPMTYSCQY (SEQ ID NO:25).
 17. A purifiedsynthetic polypeptide ligand of claim 1, wherein the 12-mer L-peptide isselected from the group consisting of VCSNMYFSCRLS (SEQ ID NO:24) andVSSNMYFSSRLS (SEQ ID NO:40).
 18. A purified synthetic polypeptide ligandof claim 1, wherein the 12-mer L-peptide is VCSNMYFSCRLS (SEQ ID NO:24).19. A purified synthetic polypeptide ligand comprising a 12-merD-peptide, fragment or homologue thereof, said 12-mer D-peptide beingthe D-form of a 12-mer L-peptide selected from the group consisting ofD-forms of 12-mer L-peptides of SEQ ID NOs:1-34, SEQ ID NOs:38-39 andSEQ ID NO:42, wherein said fragment is at least five contiguous aminoacids and wherein said homologue is at least 9/12 homologous to a 12-merD-peptide selected from said group and wherein said 12-mer D-peptide,fragment or homologue, when integrated as an N-terminal PIII fusionpeptide of an M13 phage confers an ability to bind the phage to eitherCaco-2 cell, IEC-6 cell, rat, mouse, pig or dog homogenate membranefractions, said ability being at least as great as that conferred by asimilarly integrated 12-mer peptide of SEQ ID NO:67.
 20. A purifiedsynthetic polypeptide ligand of claim 19, wherein said ligand comprisesa zinc-binding domain.
 21. A purified synthetic polypeptide ligand ofclaim 19, wherein the homologue is at least 10/12 homologous to a 12-merD-peptide.
 22. A purified synthetic polypeptide ligand of claim 19,wherein the homologue is at least 11/12 homologous to a 12-merD-peptide.
 23. A purified synthetic polypeptide ligand of claim 19,wherein said ligand comprises a 12-mer D-peptide being the D-form of a12-mer L-peptide selected from the group consisting of SEQ ID NOs:1-34,SEQ ID NOs:38-39, and SEQ ID NO:42.
 24. A purified synthetic polypeptideligand of claim 19, wherein said D-peptide is selected from the groupconsisting of SEQ ID NOs:36, 37 and 41 corresponding to the D-form of12-mer L-peptides of SEQ ID NOs:8, 25, and
 42. 25. A purified syntheticpolypeptide ligand of claim 19, wherein said ligand, consists of theD-form of an amino acid sequence selected from the group consisting ofSEQ ID NOs:1-34, SEQ ID NOs:38-39 and SEQ ID NO:42, fragment thereof orhomologue thereof.
 26. A purified nucleic acid sequence encoding apurified synthetic polypeptide ligand of claim 30, said nucleotidesequence being not more than 600 nucleotides.
 27. The purified syntheticpolypeptide ligand of claim 19, wherein said ligand consists of theD-form of a 12-mer L-peptide selected from the group consisting of SEQID NOs:1-34, SEQ ID NOs:38-39, SEQ ID NO:42.
 28. A purified syntheticpolypeptide ligand of claim 19, wherein said fragment is at least 8contiguous amino acids.
 29. A purified synthetic polypeptide ligand ofclaim 19, wherein the homologue is at least 10/12 homologous.
 30. Apurified synthetic polypeptide ligand of claim 19, wherein the homologueis at least 11/12 homologous.
 31. A purified synthetic polypeptideligand of claim 19, wherein said ligand is at most 200 amino acids inlength.
 32. A purified synthetic polypeptide ligand of claim 19, whereinsaid ligand is at least 12 amino acids in length.
 33. A purifiedsynthetic polypeptide ligand of claim 19, wherein said polypeptideligand is at least 30 amino acids in length.
 34. A purified syntheticpolypeptide ligand of claim 19, wherein the 12-mer L-peptide is selectedfrom the group consisting of LETTCASLCYPS (SEQ ID NO:8), LETTAASLCYPS(SEQ ID NO:31), LETTCASLAYPS (SEQ ID NO:32), LETTAASLAYPS (SEQ IDNO:34).
 35. A purified synthetic polypeptide ligand of claim 19, whereinsaid 12-mer L-peptide is LETTCASLCYPS (SEQ ID NO:8).
 36. A purifiedsynthetic polypeptide ligand of claim 19, wherein said 12-mer L-peptideis selected from the group consisting of VPPHPMTYSCQY (SEQ ID NO:25),VPPHPMTYSSQY (SEQ ID NO:39) and VPPHPMTYSAQY (SEQ ID NO:38).
 37. Apurified synthetic polypeptide ligand of claim 19, wherein said 12-merL-peptide is VPPHPMTYSCQY (SEQ ID NO:25).
 38. A purified syntheticpolypeptide ligand of claim 19, wherein said 12-mer L-peptide isselected from the group consisting of VCSNMYFSCRLS (SEQ ID NO:24) andVSSNMYFSSRLS (SEQ ID NO:40).
 39. A purified synthetic polypeptide ligandof claim 19, wherein said 12-mer L-peptide is VCSNMYFSCRLS (SEQ IDNO:24).
 40. A purified synthetic polypeptide ligand comprising a 12-merretro-inverted peptide, fragment or homologue thereof, said 12-merretro-inverted peptide being the retro-inverted form of a 12-merL-peptide selected from the group consisting of retro-inverted forms of12-mer L-peptides of SEQ ID NOs:1-34, SEQ ID NOs:38-39 and SEQ ID NO:42,wherein said fragment is at least five contiguous amino acids andwherein said homologue is at least 9/12 homologous to a 12-merretro-inverted peptide selected from said group and wherein said 12-merretro-inverted peptide, fragment or homologue, when integrated as anN-terminal PIII fusion peptide of an M13 phage confers an ability tobind the phage to either Caco-2 cell, IEC-6 cell, rat, mouse, pig or doghomogenate membrane fractions, said ability being at least as great asthat conferred by a similarly integrated 12-mer peptide of SEQ ID NO:67.41. A purified synthetic polypeptide ligand of claim 40, wherein saidligand comprises a zinc-binding domain.
 42. A purified syntheticpolypeptide ligand of claim 40, wherein the homologue is at least 10/12homologous to a 12-mer retro-inverted peptide.
 43. A purified syntheticpolypeptide ligand of claim 40, wherein the homologue is at least 11/12homologous to a 12-mer retro-inverted peptide.
 44. A purified syntheticpolypeptide ligand of claim 40, wherein said ligand comprises a 12-merretro-inverted peptide being the retro-inverted form of a 12-merL-peptide selected from the group consisting of SEQ ID NOs:1-34, SEQ IDNOs:38-39, and SEQ ID NO:42.
 45. A purified synthetic polypeptide ligandof claim 40, wherein said ligand, fragment or homologue, consists onlyof an amino acid sequence selected from the group consisting of SEQ IDNOs:1-34, SEQ ID NOs:38-39, and SEQ ID NO:42.
 46. A purified nucleicacid sequence encoding a purified synthetic polypeptide ligand of claim40, said nucleic acid sequence being not more than 600 nucleotides. 47.A purified synthetic polypeptide ligand of claim 40, wherein said ligandconsists of the retro-inverted form of a 12-mer L-peptide selected fromthe group consisting of SEQ ID NOs:1-34, SEQ ID NOs:38-39, and SEQ IDNO:42.
 48. A purified synthetic polypeptide ligand of claim 40, whereinsaid retro-inverted peptide is selected from the group consisting of SEQID NOs: 35 and 40 corresponding to the retro-inverted form of 12-merL-peptides SEQ ID NOs: 8 and
 25. 49. A purified synthetic polypeptideligand of claim 40, wherein said fragment is at least 8 contiguous aminoacids.
 50. A purified synthetic polypeptide ligand of claim 40, whereinthe homologue is at least 10/12 homologous to a 12-mer peptide.
 51. Apurified synthetic polypeptide ligand of claim 40, wherein said ligandis at most 200 amino acids in length.
 52. A purified syntheticpolypeptide ligand of claim 40, wherein said ligand is at least 12 aminoacids in length.
 53. The purified synthetic polypeptide ligand of claim40, wherein said polypeptide ligand is at least 30 amino acids inlength.
 54. A purified synthetic polypeptide ligand of claim. 40,wherein the 12-mer L-peptide is selected from the group consisting ofLETTCASLCYPS (SEQ ID NO:8), LETTAASLCYPS (SEQ ID NO:31), LETTCASLAYPS(SEQ ID NO:32), LETTAASLAYPS (SEQ ID NO:34).
 55. A purified syntheticpolypeptide ligand of claim 40, wherein the 12-mer L-peptide isLETTCASLCYPS (SEQ ID NO:8).
 56. A purified synthetic polypeptide ligandof claim 40, wherein the 12-mer L-peptide is selected from the groupconsisting of VPPHPMTYSCQY (SEQ ID NO:25), VPPHPMTYSSQY (SEQ ID NO:39)and VPPHPMTYSAQY (SEQ ID NO:38).
 57. A purified synthetic polypeptideligand of claim 40, wherein the 12-mer L-peptide is VPPHPMTYSCQY (SEQ IDNO:25).
 58. A purified synthetic polypeptide ligand of claim 40, whereinthe 12-mer L-peptide is selected from the group consisting ofVCSNMYFSCRLS (SEQ ID NO:24) and VSSNMYFSSRLS (SEQ ID NO:40).
 59. Apurified synthetic polypeptide ligand of claim 40, wherein the 12-merL-peptide is VCSNMYFSCRLS (SEQ ID NO:24).
 60. A purified syntheticpolypeptide ligand, said ligand comprising a L-peptide motif, aD-peptide version thereof, or a retro-inverted version thereof, saidL-peptide motif being selected from the group consisting of TPPP, PPY,PVT, LGT, NVY, HESSH (SEQ ID NO:97) and NVYTXXXXSPXP (SEQ ID NO:98),wherein said L-peptide motif, D-peptide version thereof, orretro-inverted version thereof when integrated as an N-terminal PIIIfusion peptide of an M13 phage confers an ability to bind the phage toeither Caco-2 cell, IEC-6 cell, rat, mouse, pig or dog homogenatemembrane fractions, said ability being at least as great as thatconferred by a similarly integrated 12-mer peptide of SEQ ID NO:67. 61.A purified synthetic polypeptide ligand of claim 60, said ligand notmore than 200 amino acids in length.
 62. A purified syntheticpolypeptide ligand of claim 60, said ligand not more than 50 amino acidsin length.
 63. A purified synthetic polypeptide ligand, not more than200 amino acids in length, comprising an L-peptide, fragment orhomologue thereof, said L-peptide being 6 to 12 amino acids in length,said L-peptide being selected from the group consisting of SEQ ID NOs:74through SEQ ID NO:96, wherein said fragment is at least five contiguousamino acids and wherein said homologue is at least 83% homologous to anL-peptide selected from said group wherein said L-peptide, fragment orhomologue thereof when integrated as an N-terminal PIII fusion peptideof an M13 phage confers an ability to bind the phage to either Caco-2cell, IEC-6 cell, rat, mouse, pig or dog homogenate membrane fractions,said ability being at least as great as that conferred by a similarlyintegrated 12-mer peptide of SEQ ID NO:67.
 64. A purified syntheticpolypeptide ligand of claim 63, wherein said polypeptide ligand is atmost 50 amino acids in length.
 65. A purified synthetic polypeptideligand, not more than 200 amino acids in length, comprising a D-peptide,fragment or homologue thereof, said D-peptide being 6 to 12 amino acidsin length and said D-peptide being the D-form of a L-peptide selectedfrom the group consisting of SEQ ID NOs:74 through SEQ ID NO:96, whereinsaid fragment is at least five contiguous amino acids and wherein saidhomologue is at least 83% homologous to a D-peptide selected from saidgroup and wherein said D-peptide, fragment or homologue thereof whenintegrated when integrated as an N-terminal PIII fusion peptide of anM13 phage confers an ability to bind the phage to either Caco-2 cell,IEC-6 cell, rat, mouse, pig or dog homogenate membrane fractions, saidability being at least as great as that conferred by a similarlyintegrated 12-mer peptide of SEQ ID NO:67.
 66. A purified syntheticpolypeptide ligand of claim 65, wherein said polypeptide ligand is atmost 50 amino acids in length.
 67. A purified synthetic polypeptideligand, not more than 200 amino acids in length, comprising aretro-inverted peptide, fragment or homologue thereof, saidretro-inverted peptide being 6 to 12 amino acids in length and saidretro-inverted peptide being the retro-inverted form of a L-peptideselected from the group consisting of SEQ ID NOs:74 through SEQ IDNO:96, wherein said fragment is at least five contiguous amino acids andwherein said homologue is at least 83%homologous to a retro-invertedpeptide wherein said retro-inverted peptide, fragment or homologuethereof when integrated when integrated as an N-terminal PIII fusionpeptide of an M13 phage confers an ability to bind the phage to eitherCaco-2 cell, IEC-6 cell, rat, mouse, pig or dog homogenate membranefractions, said ability being at least as great as that conferred by asimilarly integrated 12-mer peptide of SEQ ID NO:67.
 68. A purifiedsynthetic polypeptide ligand of claim 67, wherein said polypeptideligand is at most 50 amino acids in length.
 69. A purified syntheticpolypeptide ligand of claim 1, 19, 40, 60, 63, 65, or 67, wherein saidpolypeptide ligand is integrated into the protein of a phage.
 70. Theligand of claim 69, wherein said polypeptide ligand is expressed on thesurface of a phage further comprising an antigen and/or a gene encodingthe antigen also expressed on the surface.
 71. The ligand of claim 69,wherein said polypeptide ligand is expressed on the surface of abacterium further comprising an antigen and/or a gene encoding theantigen also expressed on the surface.
 72. A purified syntheticpolypeptide ligand of claim 1, 19, 40, 60, 63, 65, or 67, wherein saidpolypeptide ligand is covalently or non-covalently bound to a carrierentity comprising a pharmaceutical agent.
 73. The purified syntheticpolypeptide ligand of claim 72, wherein said carrier entity is selectedfrom the group consisting of a nanoparticle, a microparticle, aliposome, a bacterium, a phage and a virus.
 74. The purified syntheticpolypeptide ligand of claim 73, wherein said carrier entity is selectedfrom the group consisting of a nanoparticle, microparticle and aliposome.
 75. The purified synthetic polypeptide ligand of claim 74,wherein said carrier entity has a largest dimension that is in the rangeof 10 nm to 500 μm.
 76. The ligand of claim 72, wherein saidpharmaceutical agent is a drug or therapeutic agent.
 77. The ligand ofclaim 72, wherein said pharmaceutical agent is a pathogen antigen. 78.The ligand of claim 72, wherein said pharmaceutical agent is anadjuvant.
 79. The purified synthetic polypeptide ligand of claim 72,wherein said carrier entity is selected from the group consisting of aphage and a virus.
 80. A method of administering a pharmaceutical agentto an organism having intestinal epithelium, said method comprisingcontacting said intestinal epithelium with said purified syntheticpolypeptide ligand of claim 1, 19, 40, 60, 63, 65, or 67, said ligandbeing covalently, or non-covalently bound to, a carrier entity.
 81. Themethod of claim 80, wherein said organism is a mammal.
 82. The method ofclaim 81, wherein said mammal is a human.
 83. The method of claim 80,wherein said carrier entity is from the group consisting of ananoparticle, microparticle liposome, bacterial, phage and a viralcarrier.
 84. The method of claim 80, wherein said carrier entity is fromthe group consisting of a nanoparticle, microparticle and liposome. 85.The method of claim 84, wherein said carrier entity has its majordimension in the range of 10 nm to 500 μm.
 86. The method of claim 84,wherein said nanoparticle, microparticle, or liposome is loaded with apharmaceutical agent or encapsulated with a pharmaceutical agent. 87.The method of claim 80, wherein said administration is done via the oralroute.
 88. The method of claim 80 wherein said administration is donevia either a rectal, subcutaneous, intramuscular, nasal or intravenousroute.
 89. The method of claim 80, wherein said pharmaceutical agent isa vaccine.
 90. The method of claim 80, wherein said purified syntheticpolypeptide ligand is a peptide integrated into the protein of a phagewhich is coated, adsorbed or covalently bonded to a surface of a carrierthat is either a nanoparticle or microparticle.
 91. The method of claim90, wherein said phage is modified to contain DNA encoding an antigen.92. The method of claim 90, wherein said nanoparticle or microparticleis loaded with a pharmaceutical agent or encapsulated with apharmaceutical agent.
 93. A method of claim 80, wherein the purifiedsynthetic polypeptide ligand comprises a zinc-binding motif, and saidligand is contacted with said epithelium in the presence of zinc.
 94. Amethod of claim 80, wherein said carrier entity is from the groupconsisting of a phage and a viral carrier.
 95. The method of claim 94,wherein said phage comprises a targeting ligand on its surface.